PROJECT SUMMARY Cardiac contraction depends on the molecular motor myosin in sarcomeres where maintenance of contractile performance is achieved in part by the constitutive phosphorylation of myosin regulatory light chain (RLC) through the respective activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). Dilated cardiac myopathy in mice and humans results in decreased cardiac MLCK (cMLCK) expression and RLC phosphorylation while animal models with increased phosphorylation have enhanced cardiac performance with resistance to heart failure. Although it is well established that RLC phosphorylation is important for normal cardiac function, surprisingly little is known about signaling mechanisms regulating cMLCK and MLCP activities, respectively. We propose investigations on cardiac-specific signaling mechanisms for these two enzymes that affect myosin phosphorylation to elucidate cellular mechanisms for normal function as well as potential causes of decreased RLC phosphorylation in heart failure. Specific Aim 1: Having recently discovered cMLCK is phosphorylated in vivo, we plan to identify roles of specific phosphorylation sites in regulating cMLCK activity using cMLCK expression and assay systems I developed to test the hypothesis that cMLCK phosphorylation enhances its activity. These studies will be extended to intact cardiac muscle to define signaling mechanisms involved in cMLCK phosphorylation, including responsible protein kinases. Additionally, we will test the hypothesis that other forms of heart failure involve reduced RLC phosphorylation to identify potentially common signaling derangements. Specific Aim 2. Determine the roles of myosin-targeted and soluble phosphatase activities in mediating RLC dephosphorylation. Using conditional knockout models for cardiac MYPT2 and the related, ubiquitously expressed subunit MYPT1 in adult mice, we will assess the effects of specific gene ablation on cardiac function. Intact cardiac muscle trabeculae from wildtype and knockout hearts will be used to quantitatively measure contributions of distinct pools of phosphatases to maintenance of half-maximal RLC phosphorylation. Cardiomyocytes from MYPT1 and MYPT2 as well as PP1c? knockout mice will be used to identify the regulatory subunit for the soluble phosphatase. These studies will test the hypothesis that PP1c? bound and unbound to MYPT2 specifically dephosphorylates RLC, providing insights into the physiological role of myosin phosphatases in the heart. These results will also set the stage for future studies on aberrant signaling pathways that cause cardiac muscle dysfunction through effects on RLC phosphorylation.